This application to the "Innovation Grants Program for AIDS Research" proposes studies to investigate mechanisms underlying the function of the HIV-1 Nef protein. Nef plays a critical role in HIV pathogenesis, but the precise reason for its importance is not understood. Understanding of the molecular basis by which Nef contributes to HIV pathogenesis could be crucial for the development for the development of new therapeutic interventions. There is accumulating evidence that the HIV-1 Nef protein self-associates into dimers and also possibly trimers. There is also some published evidence and new data presented in our Preliminary Results that suggest that such complex formation may be regulated by Nef's association with lipid raft membrane domains. At the present time, the biological significance of Nef self-association is not clear. However, many of Nefs cellular targets are themselves regulated by oligomerization and oligomerization is a common biological regulatory mechanism. Thus it is tempting to speculate that oligomerization plays a key role in Nef function. This proposal explores the biological importance of Nef-self association as well as the mechanisms that potentially regulate it. If the oligomeric state of Nef is shown to be important, the interface between adjacent Nef molecules could become a novel target for the design of anti-HIV drugs. The specific questions that this proposal will address are: Aim #1: Does the Nef Dimer Have Functional Significance "hisaim will address two important questions. 1) Do mutations between aa 105-123 that would be expected to disrupt the Nef dimer interface, actually disrupt Nef function and dimer formation? Can dimerization and Nef function of mutant Nef molecules, be restored with compensatory mutations? Aim #2: Is Formation of the Nef Dimer Regulated by the N-terminal region of Nef and Membrane/Raft Association? Studies will be performed both in vivo and in vitro to determine if the N-teminus of Nef interacts with the Nef core to regulate accessibility and if membrane association is involved in this process.